KR101482237B1 - A pharmaceutical composition for preventing and treating autoimmune disease comprising minicircle vector expressing bispecific protein comprising anti-IL-6R and TNFR2 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of autoimmune diseases comprising a vector expressing a bispecific protein comprising anti-IL-6R and TNFR2.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for the treatment of autoimmune diseases comprising a mini-circular vector expressing a bispecific protein comprising anti-IL-6R and TNFR2, 6R and TNFR2}

The present invention relates to a pharmaceutical composition for the treatment of autoimmune diseases comprising a vector expressing a bispecific protein comprising anti-IL-6R and TNFR2.

Immunity refers to the function of protecting the body from external substances by recognizing and eliminating external antigenic substances present in the body. An autoimmune disease is a disease in which the immune system is susceptible to a part of an individual's own body, thereby destroying the body itself, resulting in a deficiency in the ability to distinguish between self and non-self. Rheumatoid arthritis, insulin- Inflammatory bowel disease, ulcerative colitis, myasthenia gravis, multiple myositis, dermatomyositis, Crohn's disease, autoimmune hematopoiesis, vasculitis syndrome, systemic lupus erythematosis, and the like.

In particular, rheumatoid arthritis, one of the autoimmune diseases, is an inflammatory autoimmune disease that is still unknown, despite the high prevalence of 21 million people, which is about 1% of the world's population. This disease causes structural malformations of the surrounding joints, and it shows persistent inflammatory synovitis that causes cartilage destruction and bone erosion. It also causes joint inflammation and pain as well as joints and surrounding tissues, and penetrates into various organs, It is accompanied by systemic organ involvement and pain, which is invaded by the eye, resulting in a tremendous loss of quality of life for the patient and disabling life like normal people.

Although autoimmune diseases have been increasing worldwide, the underlying causes of these diseases have not been sufficiently elucidated. Currently, the treatment of autoimmune diseases is aimed at regulating the autoimmune response and restoring the damaged immune function of the body. Typically, an immunosuppressant is used to suppress the immune response. Presently used immunosuppressants include prednisone, a corticosteroid drug, and cyclophosphamide, azathioprine, and tacrolimus, which are non-steroidal drugs. Recently, anti-TNF antagonists have been developed to treat various autoimmune diseases.

Tumor necrosis factor (TNF) is a multispecific cytokine that plays an important role in the inflammatory response and immune system and has been reported to play an important role in the bone destruction process. Therefore, monoclonal antibodies and recombinant proteins against TNF ligands have been developed to inhibit TNF signaling, as an autoimmune disease therapeutic agent for inhibiting the action of TNF. Infliximab, etanercept, and adalimumab have been shown to be highly efficacious in clinical use for nearly a decade as a representative treatment for autoimmune arthritis.

However, despite the high efficacy of these anti-TNF therapeutics, there are many problems that must be overcome. For example, as a side effect of treatment, the suspension of the mechanism of TNF increases the risk of infection of the fungus or germs in the immune system, resulting in an increased risk of relapsing pulmonary tuberculosis in particular. According to the Rheumatology Society, patients with rheumatoid arthritis who received anti-TNF therapies are more likely to develop skin cancer than those who received conventional treatments.

Accordingly, the present invention proposes a new concept of gene therapy agent as a therapeutic agent for autoimmune diseases which overcomes the disadvantages of safety and efficacy possessed by a single antagonist used as a conventional anti-TNF therapeutic agent.

A gene therapy method is a method of injecting a gene related to a disease into a patient's body and expressing the gene in a cell to treat a disease, wherein the injected gene is successfully transferred to the nucleus of the target cell, It is important to make them appear.

The gene transfer technology for this purpose is largely classified into a method using a virus as a carrier, a nonviral method using a synthetic phospholipid or a synthetic cationic polymer, and a physical method such as an electrotransmission method in which a gene is introduced by applying a temporary electrical stimulus to the cell membrane . Herein, the method using the viral carrier has advantages such as excellent propagation efficiency and long-term stable gene expression. However, when the administered gene permanently changes the structure of the gene in the human chromosome or has a recombinant ability, And the activation of the immune defense system of the biologic safety issues, such as the practical clinical application is subject to restrictions. The non-viral gene transfer method has the advantage of enhancing the biological stability and freely changing the chemical composition and structure of the transporter. However, the transfection efficiency and the time of expression are very inferior to those of the viral transporter, It is difficult to use for clinical use.

Therefore, the present inventors have developed a gene therapy vector system that efficiently transfers a therapeutic gene to a target cell, expresses the therapeutic gene continuously and for a long time, and is highly biologically safe. Based on this, the safety and efficacy A novel concept of an autoimmune disease gene therapy agent capable of overcoming the shortcomings of the present invention has been accomplished.

It is an object of the present invention to provide a pharmaceutical composition for the treatment of autoimmune diseases comprising a vector expressing a bispecific protein comprising anti-IL-6R and TNFR2.

The present invention relates to a pharmaceutical composition for the treatment of autoimmune diseases comprising a vector expressing a bispecific protein comprising anti-IL-6R and TNFR2.

In a preferred embodiment, the pharmaceutical composition of the present invention comprises

(a) a promoter;

A monoclonal antibody specific for IL-6R (Interleukin-6 receptor) and a TNFR2 (Tumor necrosis factor receptor type 2) protein linked to the C-terminus of the constant region of the monoclonal antibody or a fragment comprising the extracellular domain of the TNFR2 A gene encoding a bispecific protein specific for IL-6R and TNF-a; And

A gene expression cassette comprising a transcription terminator,

(b) a site-specific recombination region located outside the gene expression cassette, and

(c) does not include the cloning start point and selectable marker gene

Is a pharmaceutical composition for the prevention and treatment of autoimmune diseases including non-viral vectors.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "bispecific protein" or "bispecific antibody" refers to a protein or antibody having the same antigen binding site capable of recognizing two or more different epitopes.

The bispecific proteins herein specifically recognize TNF-a and IL-6R and are capable of double antagonistic action against them. The TNFR2 protein or the TNFR2 protein at the C-terminus of the constant region of the anti-IL-6R monoclonal antibody And has a structure of a bispecific antibody in which a fragment containing the extracellular domain of TNFR2 is bound.

Tumor necrosis factor (TNF) is a multifaceted cytokine, and TNF-α plays an important role in the inflammatory response and immune system.

In addition, interleukin (IL), a kind of cytokine, acts as a chemical signal between red blood cells. Among these, interleukin-6 (IL-6) has been found to be abnormally generated in inflammatory diseases, autoimmune diseases and tumors and has been suggested to be involved in the pathogenesis of such diseases.

In the present invention, bispecific proteins have a high binding affinity for IL-6R and TNF-a and can exhibit particularly excellent effects in the treatment of autoimmune diseases by inhibiting the action of IL-6R and TNF-a.

The term "variable region" as used herein in relation to a bispecific protein means a portion of an antibody molecule showing a large number of mutations in the sequence while performing a function of specifically binding to the antigen, and the variable region includes a complementary domain complementarity determining region (CDR), CDR1, CDR2 and CDR3. A framework region (FR) portion exists between the CDRs to support the CDR ring.

The term "complementarity determining region" as used herein also refers to an annular region involved in recognition of an antigen, and the specificity of the antibody to the antigen is determined as the sequence of the region changes.

As a preferred embodiment, the monoclonal antibody specific for IL-6R constituting the bispecific protein in the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, A heavy chain variable region comprising a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 3; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "A7" in the present specification.

In another preferred embodiment, the monoclonal antibody specific for IL-6R constituting the bispecific protein in the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, A heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 9; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

The anti-IL-6R monoclonal antibody that is identified as the heavy chain variable region and the light chain variable region as described above will be referred to as "B10" in the present specification.

In another preferred embodiment, the monoclonal antibody specific for IL-6R constituting the bispecific protein in the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, A heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 15; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 16, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 17, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 29 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 30.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "D2" in the present specification.

In another preferred embodiment, the monoclonal antibody specific for IL-6R constituting the bispecific protein in the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, A heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 21; And a light chain variable region comprising the light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, the light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 23, and the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:

More preferably, the IL-6R-specific monoclonal antibody may comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 31 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 32.

The anti-IL-6R monoclonal antibody specified as the heavy chain variable region and the light chain variable region as described above will be referred to as "F2" in the present specification.

The constant region included in the bispecific protein of the present invention may be selected from the group consisting of IgA, IgD, IgE, IgG and IgM, and preferably includes all or a part of CH1, CH2 and CH3 constant regions. Further, it is preferable, but not limited, that two Fc domains consisting of CH2 and CH3 are linked by a disulfide bond or another covalent bond.

The bispecific proteins of the invention include functional fragments of antibody molecules as well as whole antibodies. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond.

In the present invention, the TNFR2 protein constituting the bispecific protein or the fragment comprising the extracellular domain of the TNFR2 may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33.

Also preferably, the fragment comprising the extracellular domain of TNFR2 may be a polypeptide comprising Nos. 23 to 179 from the N-terminus of the amino acid sequence of SEQ ID NO: 33.

The TNFR2 protein or a fragment comprising the extracellular domain thereof contained in the bispecific protein of the present invention is linked to the C-terminus of the constant region of the monoclonal antibody specific for IL-6R.

Preferably, the bispecific protein of the invention comprises a heavy chain variable region, a heavy chain constant region, and a TNFR2 protein or a fragment comprising the extracellular region thereof, linked in the C-terminal direction from the N-terminus, respectively, of the anti-IL-6R monoclonal antibody (For example, pNATABH of the example) and a DNA comprising the light chain region of the anti-IL-6R monoclonal antibody (for example, pNATABL of the example) by co-transfection into cells and genetically recombinantly producing . The bispecific proteins thus produced will be referred to herein as "A7 / TNFR2", "B10 / TNFR2", "F2 / TNFR2", and "D2 / TNFR2", respectively.

Preferably, the TNFR2 protein or a fragment comprising its extracellular domain can be linked directly to the C-terminus of the heavy chain constant region or through a linker, such as a linker. In the present invention, when a recombinant bispecific antibody is constructed by linking an anti-IL-6R monoclonal antibody and TNFR2, a linker is inserted between a protein and a protein so as to increase the structural flexibility of the protein, Lt; / RTI > The type and number of amino acids of the linker are not limited as long as they do not decrease the overall physiological activity and can minimize the immune response, but preferably 1 to 20 amino acids, more preferably 1 to 5 amino acids .

In a specific embodiment of the present invention, an anti-IL-6R monoclonal antibody with high binding affinity for IL-6R is prepared using phage display technology, and then the antibody is immobilized on the C-terminus of the constant region of the antibody TNFR2 were fused to produce bispecific antibodies.

The term "panning ", as used in connection with phage display technology, refers to the ability to bind a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses the peptide in the coat of the phage Refers to the process of selecting only the phage expressing the peptide on the surface.

As used herein, the term "binding affinity" refers to the degree to which an antibody and an antigen form a specific binding, and the affinity of the binding is defined by the term k _a (rate constant for association of the antibody from the antibody / , k _d (dissociation constant), and K _d (k _d / k _a ).

The binding or specific binding means the binding affinity (K _d ) of 10 ^-8 mol / l or less, preferably 10 ^-9 M to 10 ^-13 mol / l. Therefore, the bispecific antibody according to the present invention specifically binds to each antigen, and its binding affinity (K _d ) is 10 ^-8 mol / l or less, preferably 10 ^-9 M to 10 ^-13 mol / l. < / RTI >

More specifically, in the present invention, an IL-6R antigen is obtained by a recombinant technique, and the IL-6R is reacted with and panned with a library phage prepared from human-derived scFv library cells having diversity, 6 monophage clones were screened. After confirming the selected monophage clones by fingerprinting, the respective sequences were analyzed to confirm the CDR regions of the variable regions (V _H and V _L ). The similarity of the variable region and the variable region of the germ line antibody group was confirmed using the Ig BLAST program of NCBI (www.ncbi.nlm.nih.gov/igblast/), and each of these monophage clones was subjected to IgG To produce a complete monoclonal antibody.

Among them, four monoclonal antibodies with excellent inhibitory effect against IL-6 were selected through STAT3 reporter analysis and TNFR2 was fused to the constant region end to produce a bispecific protein. In the present study, it was confirmed that these four bispecific proteins had strong binding to each of IL-6R and TNF-alpha, and they were named A7 / TNFR2, B10 / TNFR2, D2 / TNFR2 and F2 / TNFR2. In addition, finally, the most effective A7 / TNFR2 is provided in the present invention.

The present invention solves problems such as reduction of half-life and decrease in physiological activity due to in vivo degradation of existing protein therapeutic agents by delivering a bispecific protein in gene form to a disease site in a body and expressing the protein at a relevant site, And the treatment effect can be stably provided.

In order to solve the problems of biological stability such as immunogenicity of the viral vector system which has been used in the gene therapy method of the present invention and the problem of the delivery efficiency of the non-viral vector system, , A gene therapy vector system that can deliver therapeutic genes continuously and for a long time and has a high biological stability.

Specifically, the vector system of the present invention includes a gene expression cassette composed of only minimal essential elements such as a promoter, a therapeutic gene, and a transcription terminator. The vector system includes a selection marker such as a cloning start point and an antibiotic resistance gene It does not contain genes.

The origin of replication is mainly a bacterial sequence, which can cause an unnecessary immune response in the human body, and a selectable marker gene such as an antibiotic resistance gene can be transmitted to bacteria present in the body, There is a problem that unnecessary antimicrobial resistance may be caused. Thus, the present invention does not include a cloning start point and a selectable marker gene, thereby eliminating the problem of unnecessary immune response and antibiotic resistance. It is also possible to reduce the immune response by removing unmethylated CpG motifs derived from prokaryotic cells. In addition, since the vector of the present invention has a smaller physical size than the conventional vector, the vector of the present invention can be easily manufactured, improve the delivery efficiency, and improve the biological stability.

The vector of the present invention is characterized by being a double-stranded DNA in a circular supercoiled form.

The promoter used in the vector of the present invention may be any promoter used in the art without any particular limitation, including inducible or constitutive promoters, preferably CMV (cytomegalovirus) promoter.

The transcription terminator used in the vector of the present invention may be a terminator used in the art without any particular limitation, but may preferably contain the SV40 polyadenylation sequence.

The vector of the present invention further comprises a site specific recombination outside the gene expression cassette composed of the promoter, the gene encoding the bispecific protein as the therapeutic gene, and the transcription terminator.

The site-specific recombination region refers to a region where recombination between specific two base sequences on DNA can occur. The gene cloning method using the site-specific recombination region can be performed using a conventional method using a restriction enzyme and a ligase Which can be used as a genetic engineering method. Preferably, in the present invention, the site-specific recombination region may be an att-attached sequence derived from E. coli or bacteriophage lambda, a substrate sequence of attB or attP, or a hybrid sequence of attR or attL, It is useful for gene cloning by the attB + attP < - > attL + attR reaction in the presence of a recombinant enzyme.

In the present invention, the term " treatment and / or prophylaxis "refers to the use of a pharmaceutical composition comprising as an active ingredient a vector expressing a bispecific protein that recognizes IL-6R and TNFR2, To suppress, to mitigate, or to change advantageously. Preferably, a composition comprising a vector that expresses a bispecific protein of the invention may inhibit the action of IL-6R and TNF- [alpha] to cause autoimmune disease.

In the present invention, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, myasthenia gravis, multiple myositis, dermatomyositis, Crohn's disease, , Systemic lupus erythematosus, and the like. More preferably, it is rheumatoid arthritis.

A pharmaceutical composition comprising a vector expressing a bispecific protein that recognizes IL-6R and TNF-? Of the present invention may be formulated to additionally include a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not substantially stimulate the organism and does not interfere with the biological activity and properties of the administered ingredients. The pharmaceutically acceptable carrier in the present invention may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more components of these components, If necessary, other conventional additives such as antioxidants, buffers and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulated into injectable solutions, pills, capsules, or tablets such as aqueous solutions, suspensions, emulsions and the like.

Also, preferably, the pharmaceutical composition of the present invention may further include a filler, an excipient, a disintegrant, a binder, a lubricant, and the like. The pharmaceutical composition of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In another aspect, the invention relates to a method of treating an autoimmune disease comprising administering a pharmaceutical composition comprising a vector that expresses a bispecific protein that recognizes IL-6R and TNF-a.

The term "administering" as used herein means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the administration route of the composition of the present invention may be administered orally or parenterally in various routes &Lt; / RTI &gt;

The vector of the present invention can be produced by a number of known methods, such as electroporation, DEAE dextran, monovalent cationic liposome fusion, polyvalent cationic liposome fusion, protoplast fusion and naked DNA delivery, Or tissue. When naked DNA is delivered to a cell or tissue, the vector of the present invention containing the therapeutic gene may be blended with a sterile aqueous solution which appears to be the blood of the patient.

The therapeutic methods of the present invention include administering a pharmaceutically effective amount of a vector that expresses a bispecific protein that recognizes IL-6R and TNF- [alpha]. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. Preferably, the dosage of the pharmaceutical composition is in the range of from about 0.1 mg to about 200 mg per kg, and may be administered in single or divided doses. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The present invention specifically recognizes TNF-? And IL-6R, and efficiently transmits a bispecific protein capable of double antagonism thereto to a target cell in a gene form, and continuously and therapeutically expresses a therapeutic gene And can be applied to a gene therapy vector system having a high biological safety, it can exert an excellent effect in the treatment of autoimmune diseases.

Figure 1 shows the preparation of an IL-6R antigen for the production of an anti-IL-6R monoclonal antibody. (A) is a result of Western blotting of IL-6R secreted in the supernatant by temporarily expressing IL-6R using a pYK 604 vector in a 150 mm dish. (B) is a result of confirming the purity of IL-6R used as an antigen through chromatography, and (C) is a result of analysis using an Agilent 2100 analysis program 6R &lt; / RTI &gt;
Figure 2 shows the results of a poly-phage ELISA assay for the production of anti-IL-6R monoclonal antibodies.
Figure 3 shows the results of monophase ELISA analysis for the production of anti-IL-6R monoclonal antibodies.
Figure 4 shows fingerprinting results for a monophage clone.
FIG. 5 shows the affinity of the six different monophage clones specifically binding to IL-6R in a stepwise dilution and showing the degree of affinity to IL-6R by ELISA. The degree of affinity was changed according to the degree of dilution Both showed high binding affinity.
FIG. 6 shows phage clone screening results using IL-6R full protein among the results shown in FIG. 5. As a result, B10 has the highest binding force and shows binding strength in the order of D2, A7, A10 and A3 .
Figure 7 is a western blotted image showing that the selected monophage clones were converted to whole IgG form.
Fig. 8 shows the results of purifying IgG antibodies of monophage clones A3, A7 and A10.
FIG. 9 shows the results of purifying IgG antibodies of monophage clones B10, D2 and F2.
FIG. 10 shows the binding affinity (Kd value) of IL-6R monoclonal antibodies A3, A7, A10, B10, D2 and F2 to IL-6.
FIG. 11 shows the results of analyzing IL-6 inhibition of anti-IL-6R monoclonal antibodies A3, A7, A10, B10, D2 and F2 through STAT3 reporter analysis.
Fig. 12 shows the structure of the anti-IL-6R monoclonal antibody prepared in the present invention (left) and the structure of the bispecific antibody using the same (right).
Figure 13 shows the preparation of a fusion construct of anti-IL-6R antibody and TNFR2.
Figure 14 shows the results of confirming the successful expression of the anti-IL-6R antibody and the fusion construct of TNFR2.
Figure 15 shows the results of confirming successful purification of the anti-IL-6R antibody and the fusion construct of TNFR2.
Figure 16 shows the binding affinity (Kd value) for TNF-a of bispecific antibodies A7 / TNFR2, B10 / TNFR2, D2 / TNFR2 and F2 / TNFR2.
17 is a schematic diagram showing a manufacturing process of a mini-circle vector used in the present invention.
FIG. 18 (A) shows the results of electrophoresis after restriction enzyme digestion with respect to the parent plasmid, (B) shows the result of introducing a parent plasmid into the E. coli host so that amplification and cloning can be performed, (C) shows the results obtained by electrophoresis that a mini circle vector was prepared using Arabidopsis, and (C) shows the results obtained by electrophoresis.
FIG. 19 shows the result of confirming the expression of GFP by introducing the vector into a 293T cell line after the silver minicircle vector is constructed to express GFP.
FIG. 20 shows the result of confirming the expression of GFP in vivo by administration to a mouse after constructing the mini circle vector to express GFP.
21 (A) shows the expression of the heavy chain (HC) after inserting a DNA expressing a bispecific protein (A7 + TNFR2) into the parent plasmid, and (B) A7 + TNFR2), and then transformed into a mini-circle vector with arabinose, and then the mini-circle vector expresses a bispecific protein (A7 + TNFR2).
Figure 22 shows the results of confirming that the mini-circle vector expresses a bispecific protein (A7 + TNFR2; named Dual) through GFP expression in a 293T cell line. In each figure, Dual LD represents a low dose of a bispecific protein and Dual HD represents a high dose of a bispecific protein.
Figure 23 (A) shows the results of further confirmation of GFP expression in the 293T cell line that the mini-circle vector expresses a bispecific protein (A7 + TNFR2; named Dual). (B) shows the result of confirming the concentration of anti-IL-6R present in the culture supernatant of the cell, and (C) shows the result of confirming the concentration of the water-soluble TNFR2 present in the culture supernatant of the cell.
Figure 24 (A) shows the concentration of soluble TNFR2 in the serum of rats when tail vein injection of a mini-circle vector expressing a bispecific protein (A7 + TNFR2; named as dual-target Ab) (B) shows the result of confirming the concentration of anti-IL-6R present in the serum of the rats. WT is the result of confirming the concentration of anti-IL-6R and soluble TNFR2 in the serum of normal rats. CIA (collagen-induced arthritis) is the result of confirming the concentration of anti-IL-6R and soluble TNFR2 in the serum of collagen- Results.
25 shows a schematic diagram and injection schedule of injecting a mini-circle vector expressing a bispecific protein in the tail vein of collagen-induced arthritis (CIA) rats.
FIG. 26 is a graph showing the effect of a mini-circle vector expressing a bispecific protein (named as dual-target Ab) in a normal rat, a collagen-induced arthritis (CIA) rat, Of the swollen feet.
Figure 27 shows the incidence of rheumatoid arthritis in each of the above rats.
Fig. 28 shows the results of tissue staining of the hind paw joint of each rat.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. The anti- IL -6R Monoclonal  Preparation of antibodies

1-1. IL -6R antigen protein

The human cDNA library for IL-6R-Fc antigen was used to confirm the specificity of IL-6R by sequencing. Then, the cells were cloned into an animal cell expression vector pYK604, transfected into HEK293E cells, induced transient transfection, and cultured. After transfection, the culture supernatant was secured at intervals of 2 days, 4 days, 6 days and 9 days and purified using protein beads. The purity of IL-6R was confirmed using Protein agilent 230 kit as QC data, and gel migration was confirmed (FIG. 1).

1-2. Phage  Manufacture of library

The final OD was adjusted to 0.5-0.7 by adding 300 ml 2XYT (+ Glucose, MgCL2, CM) to the stock OD of 0.1. Helper phage was infected with MOI 20. It was held in place for 30 min at 37 ° C, followed by shaking for 10 min. The cultured solution was centrifuged at 4,500 rpm for 15 min at 4 ° C to remove the supernatant, and the media was replaced with 300 ml of 2XYT (+ MgCl2, CM, IPTG, Kan) and incubated overnight at 30 ° C. After centrifugation at 4,500 rpm for 15 min at 4 ° C, the supernatant was added to 4% PEG600 and 3% NaCl and incubated for 1 hr on ice. And centrifuged at 4 ° C at 8,500 rpm for 20 min. After removal of the supernatant, the phage was dried for 5 min and dissolved in 5 ml of cold PBS to be used for panning.

1-3. 1st ~ 3rd Panning  ( panning )

30 ug of the IL-6R full domain protein was coated on the immunotube at 4 ° C for overnight. PBS was blocked with 3 ml of 4% skim milk for 2 hours at room temperature. After that, 2 ml of phage prepared in 16% skim milk was added and then blocked for 2 hours at room temperature. 1x PBST for 10 min at 2 min. 100 mM TEA, 500 μl each for 2 minutes for 5 minutes, and neutralized with 250 μl each of 1 M Tris (pH 7.5). The elution fraction thus obtained was collected and used for titering. After leaving about 100 μl of the elution fraction, 8 ml of XL1-blue was added to the final O.D 0.5-0.7, and the cells were infected with 2 ml of the immunotube. At this time, the cells were allowed to infect at 37 ° C for 30 minutes. The infected cells were collected and centrifuged at 4,500 rpm at 4 ° C for 15 minutes. Then, the supernatant was discarded and 2 ml 2XYT (+ GLUCOSE, MgCl 2) was added to the plate. The plate was plated on a square plate and incubated at 37 ° C overnight.

As shown in the following Table 1, a human library possessed by A & RT was made into a phage form using M1 helper phage to bind to IL-6R to make an antibody against IL-6R, and the resulting product was bound to IL-6R as a primary panning output 3.1 x 10 ⁶ was obtained. The result of the first panning was converted into the phage form, bound to the IL-6R, and the second panning output was enriched to 1.69 x 10 ⁷ , and the second panning output was again The result of binding to IL-6R in phage form was enriched to 8.2 x 10 ⁸ by the third panning output.

IL-6R full 1st panning Secondary panning Third panning Input 8.5 x 10 ¹³ 2.78 x 10 ¹³ 2.3 x 10 ¹³ output 3.1 x 10 ⁶ 1.69 x 10 ⁷ 8.2 x 10 ⁸ Washing # (PBST + PBS) 7 (5 + 2) 13 (10 + 3) 23 (20 + 3)

1-4. Poly Phage ELISA  analysis

To elucidate the effect of the a-Myc, FC signal, we performed dilution of 5 each by using the supernatant obtained by phage infection. First, 100 ng / well of IL-6R, 100 ng / well of a-Myc and 100 ng / well of FC were coated on 96 well plates overnight at 4 ℃. 2% skim milk in 1X PBS was blocked with 200 μl / well at 37 ° C for 2 hours. The prepared dilution phage was added and incubated at 37 ° C for 2 hours. After washing with 1x PBST using an ELISA washer, anti-M13-HRP was bound at 1: 2000 concentration for 1 hour at 37 ° C, washed again, and read at 490nm using substrate OPD.

FIG. 2 shows the binding affinity with IL-6R by making a phage form of primary, secondary, and tertiary panned cells of the library and the presence of specificity for IL-6R as a result of the polyphage ELISA analysis. In most cases, it is hard to find any specificity in the library and 1 ^st , but after that, we can confirm that the signal is stronger from the second iterative process. Therefore, it can be confirmed that antibodies specific to IL-6R are present in the second and third orders. A-myc and Fc were used as negative controls.

1-5. Mono Phage ELISA  analysis

96-well 3- ^rd panning colonies were picked in 96-well plates (1 ml of 2XYT-glucose-MgCl2-CM) and coated overnight at 37 ° C. 100 μl of 2XYT-glucose-MgCl2-CM was incubated at 37 ° C for 3 to 4 hours at OD of 0.12. And the final OD was 0.5. helper phage was placed at 37 ° C for 10 to 30 minutes. The medium was centrifuged at 3,500 rpm for 20 minutes at 4 ° C, and the media was changed to 2XYT-IPTG-MgCl2-CM-Kan and incubated overnight at 30 ° C. The supernatant was centrifuged at 4 ° C at 3,500 rpm for 20 min, and the supernatant was used for mono-ELAISA. IL-6R full, a-Myc, and FC were coated at 4 ° C for one day in 96-well plates at 100 ng / well. After blocking at 37 ° C for 2 hours with 1 x PBS 200ul / well in 2% skim milk, the phage supernatant obtained above was once again blocked with 100ul at 37 ° C for 2 hours. 1XPBS was washed with an ELISA washer and then anti-M13-HRP was bound at 1: 2000 concentration for 50 min at 37 ° C. After further washing, substrate OPD was read at 490 nm and used.

FIG. 3 shows the results of the monophage ELISA analysis. As a result of the second panning out put and the third panning out put, the colony was selected to make 96 phage forms, and the binding affinity for IL-6R was once again confirmed. Red shades were also excluded because they also bind to Fc. Since IL-6R has Fc, it is excluded because phage binds to Fc nonspecifically.

1-6. Mono Phage  Screening and fingerprinting

The colony was picked using 1ul of the cell stock. To the colony thus obtained, 10 μl of colony PCR and 10 μl of colony PCR product were added and incubated at 37 ° C. for 2 hours in a Bst NI 0.2 μl / 20 μl reation. After that, 8% PAGE gel was prepared and electrophoresed.

6 kinds of phage forms were obtained as a result of fingerprinting of the phage form having good binding affinity to IL-6R (FIG. 4).

1-7. Selected Mono Phage  Sequence analysis of clones

DNA was extracted from the six kinds of monophage clones identified in Examples 1-6 and sequenced. Sequence analysis was performed by SolGent and ABI 3730, an automatic sequencer, was used for the analysis. As a result, CDR regions of the heavy chain (VH) and light chain (VL) of the selected antibody were identified as shown in Tables 2 and 3. Similarity between the antibody and the germ line antibody group was confirmed using NC BLI's Ig BLAST program (www.ncbi.nlm.nih.gov/igblast/). As a result, in the case of the heavy chain, the human germline sequence Homology between 87% and 96%, and between 90% and 95% homology with light chain. Also, the polypeptides used in CDR3 of the heavy chain and light chain of each human antibody were analyzed, and the sequences were found to be different from each other.

Clone name The heavy chain variable region (V _H ) CDR1 CDR2 CDR3 A7 DYAMH
(SEQ ID NO: 1) GVSWNSGTIAYVDSVKG
(SEQ ID NO: 2) DFTYFYESSGYYAFDL
(SEQ ID NO: 3) B10 DYAMF
(SEQ ID NO: 7) GINWNGNGIGYGDSVRG
(SEQ ID NO: 8) PSLYGGNSEFDL
(SEQ ID NO: 9) D2 NYAIN
(SEQ ID NO: 13) RIIPMLGTSDYAEKFQG
(SEQ ID NO: 14) GPRYYGTDSYYLEK
(SEQ ID NO: 15) F2 NYYMH
(SEQ ID NO: 19) IINPSGGNTGYAQKFQG
(SEQ ID NO: 20) GLPWGENGLDV
(SEQ ID NO: 21) A3 EYAFH
(SEQ ID NO: 38) DFTPAFGEGDTAQKFQG
(SEQ ID NO: 39) ETASTY
(SEQ ID NO: 40) A10 DYAMH
(SEQ ID NO: 44) VISGDDGTTHYADSVKG
(SEQ ID NO: 45) DMYPYGDYGYLQH
(SEQ ID NO: 46)

Clone name The light chain variable region (V _L ) CDR1 CDR2 CDR3 A7 TGTNSNIGAGYDVH
(SEQ ID NO: 4) GNTNRPS
(SEQ ID NO: 5) QSFDSSLT
(SEQ ID NO: 6) B10 TGPTIGAGYDVH
(SEQ ID NO: 10) GNLNRPS
(SEQ ID NO: 11) HTYDSSLS
(SEQ ID NO: 12) D2 TGPTIGAGYDVH
(SEQ ID NO: 16) GNLNRPS
(SEQ ID NO: 17) HTYDSSLS
(SEQ ID NO: 18) F2 TGSSSNIGAGYDVH
(SEQ ID NO: 22) GDSDRPS
(SEQ ID NO: 23) QSYDSSLS
(SEQ ID NO: 24) A3 TGPTIGAGYDVH
(SEQ ID NO: 41) GNLNRPS
(SEQ ID NO: 42) HTYDSSLS
(SEQ ID NO: 43) A10 RASQGISSYLA
(SEQ ID NO: 47) AASTLQS
(SEQ ID NO: 48) QQLNTYPL
(SEQ ID NO: 49)

Clone name V _H Homology V _L Homology group IL-6R full a-myc Fc Ratio A03 VH1-69 252/288
(87.5%) V1-13 265/293
(90.4%) One 2.1076 0.2999 0.0418 7.027675892 A07 VH3-9 279/293
(95.2%) V1-13 279/293
(95.2%) 2 2.4774 0.0797 0.0421 31.08406524 A10 VH3-43 270/295
(91.5%) L8 271/286
(94.8%) 3 1.435 0.8478 0.095 1.692616183 6B10 VH3-9 260/287
(90.6%) V1-13 265/293
(90.4%) 4 2.0979 0.1125 0.0406 18.648 D02 VH1-69 267/289
(92.4%) V1-13 265/293
(90.4%) 5 2.3382 0.3031 0.0415 7.714285714 F02 VH1-46 286/295
(96.9%) V1-13 277/292
(94.9%) 6 2.8567 0.2869 0.0405 9.957127919

1-8. IL-6R full , IL-6R FN3 - FN3 Ranking ELISA

FIG. 5 shows ELISA showing the degree of affinity to IL-6R by stepwise dilution of six different monophage clones specifically binding to IL-6R. The ELISA showed a change in binding force according to the degree of dilution Both showed high binding affinity.

FIG. 6 shows phage clone screening results using IL-6R full protein among the results shown in FIG. 5. As a result, B10 has the highest binding force and shows binding strength in the order of D2, A7, A10 and A3 . F2 showed the lowest bond strength.

1-9. all IgG  Conversion analysis

After conversion of the six monophage clones into whole IgG form, Western blot analysis confirmed whether the assemble was successful in mammalian cells. A3 was weakly assemble, but all five were well assimilated and expression levels were high (Fig. 7).

1-10. term IL -6R Monoclonal  Purification of antibodies

The purified purity and gel migration were confirmed using Agilent 230Kit as QC data for each of the 6 antibodies (FIGS. 8 and 9).

Example  2. The anti- IL -6R Monoclonal  Binding Affinity Analysis of Antibodies

2-1. term IL -6R Monoclonal  Antibody Kd  Measure value

IL-6R at 100 ng / well for one day. Each well was blocked with 2% skim milk (200 ul) at 37 ° C for 2 h. Dilution was performed twice at 50 nm, and the mixture was incubated at 37 ° C for 2 hours to bind 100ul each. After washing 3 times with 1xPBST, anti-Fab-HRP was bound at a concentration of 1: 4000 in 100ul at 37 ° C for 1 hour. After washing 3 times with 1xPBST, OPD 1tablet and 6ul H ₂ O ₂ were added to 10ml of PC buffer. After treating with 100ul at room temperature for 7min, the reaction was stopped with 50ul of NH ₂ SO ₄ and read at 490nM.

Table 5 below shows the results of measurement of the Kd value of each antibody according to the dilution concentration of IL-6R, and FIG. These results indicate that A3, A7 and F2 exhibit very high binding affinity for very small amounts (0.02441 nM) of IL-6R protein.

Clone name 50 25 12.5 6.25 3.125 1.5625 0.78125 0.39063 0.19531 0.09766 0.04883 0.02441 kd value A3 2.781 2.2787 1.8467 1.4486 1.1317 0.857 0.6503 0.5904 0.5249 0.4996 0.4894 0.5156 3.5x10 ^-9
(R ² : 0.83) A7 3.0994 2.8431 2.5795 2.1338 1.6383 1.1682 0.7819 0.6175 0.5464 0.4973 0.4983 0.5145 2.3x10 ^-9
(R ² : 0.93) A10 2.5724 2.2591 1.8109 1.4725 1.1259 0.8664 0.6497 0.5731 0.5022 0.4599 0.4693 0.4952 2.93x10 ^-9
(R ² : 0.85) B10 2.0937 1.7787 1.3939 1.0362 0.761 0.6069 0.5 0.4762 0.4289 0.4619 0.4508 0.4679 4.3x10 ^-9
(R ² : 0.75) D2 2.3833 1.9404 1.4694 0.9809 0.7194 0.5843 0.4786 0.4732 0.4688 0.4498 0.4514 0.4486 7.5x10 ^-9
(R ² : 0.8) F2 2.9947 2.8868 2.9209 2.7102 2.7158 2.433 1.9001 1.4102 1.0056 0.7428 0.5836 0.535 3.5x10 ^-10
(R ² : 0.98)

2-2. STAT3 - by reporter analysis IL -6 Inhibition  Test

A STAT3-reporter assay was performed to confirm the function of the anti-IL-6R monoclonal antibody produced. STAT3 was expressed in a cell line expressing the reporter gene with lucifrerase attached thereto. IL-6 was added to the cell line at a dose of 50 ng / well in all conditions except for the control group. After that, anti-IL-6R prepared according to each condition was treated with 0.001 ug, 0.01 ug, 0.1 ug and 1 ug / well, respectively, and the signal induced by IL-6 was detected by anti-IL-6R Was measured. The prepared cells were treated with each cytokine and antibody and then incubated for 24 hours. After that, lucifease substrate was treated with each cell to induce color development, and the degree of expression was confirmed by measuring the color.

As a result, anti-IL-6R antibodies of A7, B10, D2 and F2 showed the effect of inhibiting IL6 signal. Among them, A7 and F2 were superior to the other inhibitory effects (Fig. 11), and thus it was decided to fuse these antibodies with TNFR2.

Example  3. Double specific  Antibody ( bi - specific antibody )

A bispecific antibody capable of specifically recognizing IL-6R and TNFR2 was prepared using the anti-IL-6R monoclonal antibody prepared in Example 2 above. Fig. 12 shows the structure of the anti-IL-6R monoclonal antibody prepared in the present invention (left) and the structure of the bispecific antibody using the same (right). The specific production process is as follows.

Specifically, anti-IL-6R / TNFR2 (designated as A7 / TNFR2, B10 / TNFR2, F2 / TNFR2 and D2 / TNFR2, respectively) , F2, and D2) was fused to the extracellular domain of Opti-TNFR2 at the C-term end. The insert was inserted into pNATABH, a heavy chain (HC) mammalian expression vector to obtain a specific antibody protein. (SEQ ID NO: 34), B10 heavy chain + TNFR2 (SEQ ID NO: 35), F2 heavy chain + TNFR2 (SEQ ID NO: 36) and D2 heavy chain + TNFR2 (SEQ ID NO: 37) (# R033S, Enzynomics, korea) and NheI (# R016M, Enzynomics, korea) and then loaded on the gel of Agarose (# 9002-18-0, BIO BASIC INC, USA) # 1014876, QIAGEN, USA) to obtain an insert to be inserted into the vector. Ligation was performed using T4 DNA ligase (# M001S, Enzynomics, Korea). The restriction enzyme reaction and the ligation reaction were carried out in the same manner under the following conditions.

The restriction enzyme reaction was performed by mixing 1 μl SfiI (10 units), 1 μl NheI and 5 μl 10 × reaction buffer, sterilized distilled water to make a total volume of 50 μl, reacted for 6 hours at 37 ° C in a hot water bath, Separation purification was performed from gel. Ligation was carried out by incubating the 50ng vector with 1ul Ligase (10 units) and 1ul 10x reaction buffer at 16 ° C for more than 16hr.

The obtained ligates were transformed into E. coli cells, and colonies were inserted into the pNATABH heavy chain expression vector by sequencing to obtain a clone, followed by midi prep (# 740 410.100, MACHERY-NAGEL, Germany) Respectively.

The A7 light chain, the B10 light chain, the F2 light chain, and the D2 light chain were amplified by the PCR method to allow insertion into the light chain (HC) mammalian expression vector pNATABL. Then, SfiI (# R033S, Enzynomics, Korea), BglII (# R010M, Enzynomics, korea) and then loaded on the agarose gel (# 9002-18-0, BIO BASIC INC, USA) QIAGEN, USA) to obtain an insert to be inserted into the vector. Ligation was performed using T4 DNA ligase (# M001S, Enzynomics, Korea). The restriction enzyme reaction and the ligation reaction were carried out in the same manner under the following conditions.

The restriction enzyme reaction was performed by mixing 1 ul SfiI (10 units), 1 ul BglII and 5 ul 10x reaction buffer, adding sterilized distilled water to make the total volume 50 μl, reacting for 6 hours at 37 ° C in a hot water bath, Separation purification was performed from gel. Ligation was carried out by incubating the 50ng vector with 1ul Ligase (10 units) and 1ul 10x reaction buffer at 16 ° C for more than 16hr.

The obtained ligates were transformed into E. coli cells, and the clones were inserted into the pNATABL light chain expression vector by sequencing the resulting colonies. The midi prep (# 740 410.100, MACHERY-NAGEL, Germany) was performed Respectively.

To obtain the bispecific antibody protein, anti-IL-6R / TNFR2 expression was confirmed by co-transfection in 293E mammalian cell with 3: 7 ratio of heavy chain vector and light chain vector, respectively. The fusion antibodies Anti-IL-6R (A7, B10, F2, D2) / TNFR2 proteins were obtained.

Example  4. Double specific  Binding Affinity Analysis of Antibodies

(# C001-1MG, enzynomics, Korea) per well using an ELISA plate (# 439454, Nunc, Denmark). The coating was performed overnight at 4 &lt; 0 &gt; C. To the coated ELISA plate, a solution containing 4% skim milk (# 232100, Difco, France) in 200 μl of PBS was added and blocking was performed for 1 hour at room temperature (blocking: 4% skim milk / 1xPBS ). After blocking, the blocking buffer was removed from the ELISA plate, and 100 μl of each purified protein was added to 100 μl of a 1% skim milk solution in PBS, which was diluted to 1/4 dilution (serial dilution) and allowed to react at room temperature for approximately 2 hours. The plate was then washed five times with 200 μl PBST and 2 μl of secondary antibody anti-Human Fc-HRP (# 31413, Thermo, USA) was added to 4 ml of 1% skim milk After mixing with PBS, 200 쨉 l per well of an ELISA plate was added, followed by reaction at room temperature for 1 hour. After the reaction, the secondary antibody on the ELISA plate was removed and then washed five times with 200 μl of PBS. 10 μl of hydrogen peroxide solution (H2O2, # H1009-100ML, Sigma, USA) and 10 ml of PC buffer Citric acid monohydrate, 7.3 g sodium phosphate / L (pH 5.0)) and OPD purification (# P8787-100TAB Sigma USA) are mixed and added to a total volume of 100 μl. After the reaction solution was added, the reaction was allowed to proceed for 10 minutes at room temperature in a dark state. Then, 50 μl of STOP buffer for stopping the reaction was added to terminate the reaction, and the reaction was terminated by an ELISA reader at a wavelength of 490 nm The Kd value was measured. The TNF-α ligand showed a low Kd value with each protein, confirming the high binding force. By Anti-IL-6R / TNFR2 antibody even for four kinds when the check binding capacity with TNF-a alone as the TNFR2 ^{7.8x10 -11 M, 7.2x10 -10 M,} 4.3x10 -10 M, 6x10 -10 M The results are shown in Fig.

Antibody antigen
IL-6R antigen
TNF-a A7 2.3 nM - B10 4.3 nM - D2 7.5 nM - F2 0.35 nM - TNFR2 - 0.068 nM A7 / TNFR2 2.3 nM 0.078 nM B10 / TNFR2 4.3 nM 0.72 nM D2 / TNFR2 7.5 nM 0.60 nM F2 / TNFR2 0.35 nM 0.43 nM

Example  5. Manufacture of Mini Circle Vector

5-1. Of DNA  Amplification

1 μg of the DNA sample (GFP or A7 + TNFR2) to be amplified was mixed with 100 μl of competent cells and incubated at 4 ° C. for 20 minutes. Then, 100 μl of competent cell (DH5a) was dissolved at 4 ° C. for 5 minutes. Lt; 0 &gt; C for 45 seconds. Lt; RTI ID = 0.0 &gt; 4 C &lt; / RTI &gt; for 2 minutes. Thereafter, 500 μl of LB (lysogeny broth) liquid medium was added thereto, followed by incubation at 37 ° C. for 1 hour with stirring at 180 rpm. After centrifugation at 5000 rpm for 30 seconds, 500 쨉 l of the supernatant was removed. The remaining cells were plated on an LB plate containing kanamycin and then incubated in a 37 ° C incubator for one day. One colony was picked from a LB plate streaked overnight in 5 ml LB medium containing kanamycin and incubated for 7 to 8 hours with stirring at 180 rpm. 1 ml of 5 ml LB medium previously incubated with 200 ml LB medium containing kanamycin was added, followed by incubation with stirring at 180 rpm for about 15 hours. After the incubation was completed, the mixture was centrifuged at 4,000 rpm for 15 minutes at 9000 rpm, and the supernatant was discarded. DNA was extracted using the Macherey-nagel Nucleobond Xtra Midi kit (Cat # REF 740410.50).

5-2. A parental plasmid ZYCY10P3S2T  E. coli Transformation into

100 ㎕ of competent cell (ZYCY10P3S2T) was dissolved at 4 째 C, 1 DNA of DNA was mixed with competent cells, and incubated at 4 째 C for 30 minutes. After applying a thermal shock for 30 seconds at 42 캜, the ice was subjected to cold shock for 2 minutes. At the end of the low-temperature impact, 0.5 ml of SOC medium was added, and incubation was carried out for 1 hour while stirring at 225 rpm at 37 占 폚. Then, 100 占 퐇 was taken and plated on an LB medium plate containing kanamycin. Plates were incubated overnight in a 37 &lt; 0 &gt; C incubator.

5-3. Mini Circle Vector ( minicircle DNA ) Production

Colonies were picked from 5 ml of LB medium containing kanamycin in a plate into which DNA was introduced into ZYCY10P3S2T competent cells. The LB medium containing the cells was incubated at 37 DEG C and 200 rpm for 8 hours. 100 μl of the incubated LB medium was added to 200 ml of TB (terrific broth) medium containing kanamycin, and TB buffer was incubated at 37 ° C and 200 rpm overnight (15 hours). On the next day, the absorbance at OD600 was measured. When the value was between 4 and 5, a minicircle induction mix was prepared by adding 8 ml 1N NaOH and 200 l 20% L-arabinose to 200 ml LB medium And then mixed with the incubated TB medium. And incubated at 30 DEG C with stirring at 200 rpm for 5 hours. After the incubation, centrifugation was carried out at 4 DEG C at 9000 rpm for 15 minutes, and the supernatant was discarded except for the pellet. DNA was extracted using a Nucleobond Xtra Midi kit (Macherey-nagel, Cat #REF 740410.50.).

5-4. Mini circle with restriction enzyme DNA Confirm the creation of

To confirm that the mini-circle DNA was properly generated, restriction enzyme reaction was performed with restriction enzymes BamHI and XbaI (enzynomics). The prepared mini-circle DNA was mixed with restriction enzyme buffer II, BSA, and third distilled water in an appropriate amount, followed by incubation at 37 ° C for 30 minutes. The incubated samples were electrophoresed on 0.8% agarose gel along with DNA samples without restriction enzyme treatment.

FIG. 18 (A) shows the results of electrophoresis after restriction enzyme digestion with respect to the parent plasmid, (B) shows the result of introducing a parent plasmid into the E. coli host so that amplification and cloning can be performed, (C) shows the results obtained by electrophoresis that a mini circle vector was prepared using Arabidopsis, and (C) shows the results obtained by electrophoresis.

Example  6. Through mini circle vector GFP  Identification of protein expression

6-1. Identification from cell lines ( in vitro )

To confirm that the mini circle DNA generated in Example 5 correctly induced protein synthesis, mini circle DNA with GFP inserted was transfected into 293T cells. On the day before transfection, 3 x 10 &lt; ⁴ &gt; of 293T cells were plated in 96 well plates with 100 [mu] l of DMEM medium containing 7.5% FBS but no antibiotic. 0.2 μg of Mini Circle DNA was mixed with 25 μl of opti-MEM1, and 0.5 μl of lipofectamine 2000 (Invitrogen) and 25 μl of opti-MEM were mixed, followed by incubation at room temperature for 5 minutes. Mini-Circle DNA and Lipofectamine 2000 were mixed together and incubated at room temperature for 20 minutes. Then, the mixture was sprayed on 293T cells, incubated at 37 ° C for 4 hours, and changed into growth medium. Expression was confirmed 24 hours later.

FIG. 19 shows the result of confirming expression of GFP by introducing the mini circle vector into a 293T cell line after constructing to express GFP.

6-2. In rats  Confirm ( in vivo )

Mini circle DNA was introduced into rats to confirm that the mini circle DNA generated in Example 5 expressed proteins in the body. Female mice 6-7 weeks old were used with DBA1 / J (Orient Bio). 7-14 mu g of GFP-infected Mini Circle DNA was mixed with 1.5-1.8 ml of saline corresponding to 10% of the body weight of the mice, Intravenously into the tail.

FIG. 20 shows the result of confirming expression of GFP in vivo by administration to a mouse after constructing the mini-circle vector to express GFP.

Example  7. Through mini circle vector Double specific  Identification of Protein Expression

7-1. Mini circle with restriction enzyme DNA Confirm the creation of

A mini-circle vector into which a DNA expressing a heavy chain of a bispecific protein (A7 + TNFR2 HC) was inserted was prepared according to the methods of Examples <5-1> to <5-4> It was confirmed that DNA was generated.

21 (A) shows the expression of the heavy chain (HC) after inserting a DNA expressing a bispecific protein (A7 + TNFR2) into the parent plasmid, and (B) A7 + TNFR2), and then transformed into a mini-circle vector with arabinose, and then the mini-circle vector expresses a bispecific protein (A7 + TNFR2).

7-2. Identification from cell lines ( in vitro )

To confirm that the Mini Circle DNA generated in Example 5 expresses the heavy chain of the bispecific protein (A7 + TNFR2 HC), DNA encoding GFP and the heavy chain of the bispecific protein (A7 + TNFR2 HC) The mini-circle DNA was transfected into 293T cells and its expression was confirmed by the method of Example < 6-1 >.

Figure 22 shows the results of confirming that the mini-circle vector expresses a bispecific protein (named Dual) through GFP expression in a 293T cell line. In each figure, Dual LD represents a low dose of a bispecific protein and Dual HD represents a high dose of a bispecific protein.

7-3. By ELISA  Confirmation of expression

In order to confirm the expression of the bispecific protein, 293T cells were transfected with mini-circle DNA, and culture supernatant was obtained therefrom, and ELISA for anti-IL-6R was performed. 100 μl of anti-hIL-6R antibody (1 μg / ml) was added to a 96-well corning coaster 9018 plate and kept at 4 ° C. overnight. After washing five times with wash buffer, 100 μl of human sIL-6R was added at 1 μg / ml and incubated at room temperature for 2 hours. After washing again, the 1x assay diluent was treated with 200 쨉 l per well and incubated at room temperature for 1 hour. After washing five times with wash buffer, tociluzumab was diluted to a standard of 100 pg / ml in 1x assay diluent, and the remaining samples were diluted with a certain amount of serum samples, and then treated with 100 μl each well. Lt; / RTI &gt; After washing five times, anti-hIgG-HRP as a detection antibody was diluted at a ratio of 1: 10,000, and the mixture was incubated at room temperature. After washing 7 times, TMB substrate solution was added and incubated at room temperature for 15 minutes. 50 [mu] l of the termination solution was treated, and the absorbance at 450 nm was measured.

In order to confirm the expression of the bispecific protein, 293T cells were transfected with mini-circle DNA, and culture supernatant was obtained therefrom. ELISA was performed on soluble TNFR2 (soluble TNFR2, sTNFR2) kit (Ready-to-use sandwich ELISA, eBioscience) protocol. Briefly, a culture supernatant sample and a sample for a standard curve were added to each well on a plate already coated with antibody against sTNFR2, and incubated at room temperature for 2 hours. Thereafter, the washing procedure was carried out four times. Then, 100 μl of the TMB substrate solution was treated and incubated at room temperature for 10 minutes. Then, 100 mu l of the reaction stop solution was added and the absorbance was measured at 450 nm.

23 (A) shows a result of confirming again that the mini-circle vector expresses the heavy chain of the bispecific protein (A7 + TNFR2 HC) through GFP expression in the 293T cell line, and (B) IL-6R present in the supernatant, and (C) shows the result of confirming the concentration of the water-soluble TNFR2 present in the culture supernatant of the cell.

7-4. In rats  Confirm ( in vivo )

Mini-circle DNA was introduced into rats to confirm that mini-circle DNA also expresses a bispecific protein in the body. Female mice 6-7 weeks old with DBA1 / J (Orient Biotechnology) were used, and 7-14 μg of Mini Circle DNA expressing A7 + TNFR2 bispecific protein was injected via a tail vein injection in a mouse by hydrodynamic transfer .

The serum of the rats was collected and the concentration of the protein present in the serum was measured in the manner described in <7-3>. 24 (A) shows the result of confirming the concentration of anti-IL-6R present in the serum of the rats, and (B) shows the result of confirming the concentration of the soluble TNFR2 present in the serum of the rats. These results implied that both IL-6R and sTNFR2 were detected in the serum of rats, so that the presence of a bispecific protein including IL-6R and sTNFR2 in the serum of the rats can be indirectly confirmed.

Experimental Example  One. Rheumatism  In an arthritis animal model Double specific  Therapeutic effect of mini circle vector expressing protein

1-1. Check the degree of swelling of the foot

An animal model of rheumatoid arthritis was constructed by using mouse monoclonal anti-type II collagen 5 clone antibody cocktail kit (chondrex 53010) in 7-week-old DBA1 / J rats as manual. 6 mg of 5-clone cocktail was intraperitoneally administered, and 3 days later, 25-50 μg of LPS was intraperitoneally administered to construct a disease model. The experiment was carried out by administering a mini-circle vector expressing the A7 + TNFR2 bispecific protein in an animal model of collagen-induced arthritis (CIA) on the stomach (FIG. 25).

The degree of paw swelling of each animal was measured and shown in Fig. The swelling of each joint in the rat was scored as 0 ~ 4 points, and the scores were added to the values. The higher the score, the more severe the disease. In the case of the disease group (CIA), rheumatoid arthritis was induced and swelling of the foot was increased compared to the normal group. In addition, it was confirmed that when a mini-circle vector expressing the A7 + TNFR2 bispecific protein was administered, the swelling of joints was alleviated to prevent arthritis.

1-2. Rheumatism  arthritis Incidence rate  Confirm

FIG. 27 is a graph showing the incidence rate. In the test group of Experimental Example 1-1, the incidence was lower in the group to which A7 + TNFR2 bispecific protein-expressing mini-circle vector was administered than in the CIA model, Can be confirmed.

1-3. Tissue staining

At the time of completion of the experiment of Experimental Example 1-1, rats were sacrificed and the biceps of the hind foot was separated to perform tissue staining. In the case of H & E staining, Xylene Ⅰ and xylene Ⅱ were passed through a deparaffin process for 15 minutes, followed by 5 minutes each in the order of 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol (hydration function). And washed in tap water for 5 minutes. After staining with Harris hematoxylin (Sigma-Aldrich HHS32) for 10 minutes, it was washed thoroughly with tap water, dipped 2-3 times in 1% HCl and 2-3 times in 0.2% ammonia water. The cells were stained with Eosin (Sigma-Aldrich HT110132) for 1 minute and 30 seconds and then washed. 70% ethanol, 80% ethanol, 90% ethanol and 100% ethanol (dehydration dehydration) were sequentially passed through the column, and the column was immersed in Xylene. In the case of Safranin O staining, the same procedure was performed and only the dye was used. At this time, the cells were stained with Weigert's iron hematoxylin (Sigma-Aldrich HT1079-1SET) and Fast green (FCF, Sigma-Aldrich F7252-5G) for 5 minutes. After dipping 2-3 times in 1% acetic acid and staining for 5 minutes with Safranin® (Sigma-Aldrich S2255-25G), the cells were washed in tap water. The same sealing process was then performed.

The results are shown in Fig. In the case of the disease group (CIA), peanut-shaped tasal bone is destroyed, but it can be confirmed that it is restored by the administration of mini circle vector expressing A7 + TNFR2 bispecific protein. In addition, in the case of the disease group (CIA), cartilage in the joints was not stained due to destruction, but it was stained again when the mini-circle vector expressing the A7 + TNFR2 bispecific protein inhibited cartilage destruction. .

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A pharmaceutical composition for prevention and treating          autoimmune disease comprising minicircle vector expressing          bispecific protein comprising anti IL-6R and TNFR2 <130> DPP20131628KR <150> KR10-2012-0050444 <151> 2012-05-11 <160> 49 <170> Kopatentin 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A7 heavy chain variable region <400> 1 Asp Tyr Ala Met His   1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A7 heavy chain variable region <400> 2 Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr Val Asp Ser Val Lys   1 5 10 15 Gly     <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A7 heavy chain variable region <400> 3 Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly Tyr Tyr Ala Phe Asp Leu   1 5 10 15 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A7 light chain variable region <400> 4 Thr Gly Thr Asn Ser Asn Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A7 light chain variable region <400> 5 Gly Asn Thr Asn Arg Pro Ser   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A7 light chain variable region <400> 6 Gln Ser Phe Asp Ser Ser Leu Thr   1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of B10 heavy chain variable region <400> 7 Asp Tyr Ala Met Phe   1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of B10 heavy chain variable region <400> 8 Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr Gly Asp Ser Val Arg   1 5 10 15 Gly     <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of B10 heavy chain variable region <400> 9 Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe Asp Leu   1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of B10 light chain variable region <400> 10 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of B10 light chain variable region <400> 11 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of B10 light chain variable region <400> 12 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of D2 heavy chain variable region <400> 13 Asn Tyr Ala Ile Asn   1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of D2 heavy chain variable region <400> 14 Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr Ala Glu Lys Phe Gln   1 5 10 15 Gly     <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of D2 heavy chain variable region <400> 15 Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr Tyr Leu Glu Lys   1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of D2 light chain variable region <400> 16 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of D2 light chain variable region <400> 17 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of D2 light chain variable region <400> 18 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of F2 heavy chain variable region <400> 19 Asn Tyr Tyr Met His   1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of F2 heavy chain variable region <400> 20 Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of F2 heavy chain variable region <400> 21 Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp Val   1 5 10 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of F2 light chain variable region <400> 22 Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of F2 light chain variable region <400> 23 Gly Asp Ser Asp Arg Pro Ser   1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of F2 light chain variable region <400> 24 Gln Ser Tyr Asp Ser Ser Leu Ser   1 5 <210> 25 <211> 129 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > A7 heavy chain variable region <400> 25 Asp Val His Ser Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Thr Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Lys              20 25 30 Phe Glu Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ser Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr      50 55 60 Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys  65 70 75 80 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly             100 105 110 Tyr Tyr Ala Phe Asp Leu Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 125 Ser     <210> 26 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> A7 light chain variable region <400> 26 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Thr Asn Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Val Pro Gly Thr          35 40 45 Thr Pro Lys Leu Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Gly Gly Asp Tyr Tyr Cys Gln Ser                  85 90 95 Phe Asp Ser Ser Leu Thr Gly Ser Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> B10 heavy chain variable region <400> 27 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Ile              20 25 30 Phe Asp Asp Tyr Ala Met Phe Trp Val Arg Gln Val Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ala Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr      50 55 60 Gly Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Arg  65 70 75 80 Asn Ser Leu Asp Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe             100 105 110 Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 28 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> B10 light chain variable region <400> 28 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 29 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> D2 heavy chain variable region <400> 29 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Gly Ser Gly Asp Thr              20 25 30 Phe Pro Asn Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Pro Glu Trp Met Gly Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr      50 55 60 Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr  65 70 75 80 Asn Thr Ala Tyr Met Gly Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Val Lys Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr             100 105 110 Tyr Leu Glu Lys Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 30 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> D2 light chain variable region <400> 30 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Pro Thr Ile Gly              20 25 30 Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro          35 40 45 Lys Leu Leu Ile Tyr Gly Asn Leu Asn Arg Pro Ser Gly Val Pro Asp      50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr  65 70 75 80 Asp Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys His Thr Tyr Asp                  85 90 95 Ser Ser Leu Ser Gly Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 31 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> F2 heavy chain variable region <400> 31 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr              20 25 30 Phe Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr      50 55 60 Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Met Ser Thr  65 70 75 80 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 32 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> F2 light chain variable region <400> 32 Asp Val His Ser Gln Leu Val Leu Thr Gln Pro Ser Ser Val Ser Gly   1 5 10 15 Ala Pro Gly Gln Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn              20 25 30 Ile Gly Ala Gly Tyr Asp Val His His Trp Tyr Gln Gln Leu Pro Gly Thr          35 40 45 Ala Pro Lys Val Leu Ile Tyr Gly Asp Ser Asp Arg Pro Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala  65 70 75 80 Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser                  85 90 95 Tyr Asp Ser Ser Leu Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val             100 105 110 Thr Val Leu         115 <210> 33 <211> 230 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) <223> TNFR2 <400> 33 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser   1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys              20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr          35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu      50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser  65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys                  85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys             100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala         115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro     130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala                 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Ser Ala Pro Gly Ala Val             180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr         195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly     210 215 220 Pro Ser Pro Pro Ala Glu 225 230 <210> 34 <211> 637 <212> PRT <213> Artificial Sequence <220> <223> A7 heavy chain + TNFR2 <400> 34 Asp Val His Ser Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Thr Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Lys              20 25 30 Phe Glu Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ser Gly Val Ser Trp Asn Ser Gly Thr Ile Ala Tyr      50 55 60 Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys  65 70 75 80 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Asp Phe Thr Tyr Phe Tyr Glu Ser Ser Gly             100 105 110 Tyr Tyr Ala Phe Asp Leu Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 125 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser     130 135 140 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr                 165 170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr             180 185 190 Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln         195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp     210 215 220 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro                 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr             260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn         275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg     290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser                 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys             340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp         355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe     370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe                 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly             420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr         435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala     450 455 460 Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu 465 470 475 480 Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly                 485 490 495 Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp             500 505 510 Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu         515 520 525 Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln     530 535 540 Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp 545 550 555 560 Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu                 565 570 575 Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr             580 585 590 Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr         595 600 605 Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val     610 615 620 Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 35 <211> 633 <212> PRT <213> Artificial Sequence <220> <223> B10 heavy chain + TNFR2 <400> 35 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Ile              20 25 30 Phe Asp Asp Tyr Ala Met Phe Trp Val Arg Gln Val Pro Gly Lys Gly          35 40 45 Leu Glu Trp Val Ala Gly Ile Asn Trp Asn Gly Asn Gly Ile Gly Tyr      50 55 60 Gly Asp Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Arg  65 70 75 80 Asn Ser Leu Asp Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Pro Ser Leu Tyr Gly Gly Asn Ser Glu Phe             100 105 110 Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser     130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys         195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys                 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val             260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp         275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr     290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu                 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg             340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys         355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp     370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser                 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser             420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser         435 440 445 Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr     450 455 460 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 465 470 475 480 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys                 485 490 495 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp             500 505 510 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys         515 520 525 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg     530 535 540 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 545 550 555 560 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg                 565 570 575 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val             580 585 590 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr         595 600 605 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly     610 615 620 Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 36 <211> 635 <212> PRT <213> Artificial Sequence <220> <223> F2 heavy chain + TNFR2 <400> 36 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Gly Ser Gly Asp Thr              20 25 30 Phe Pro Asn Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Pro Glu Trp Met Gly Arg Ile Ile Pro Met Leu Gly Thr Ser Asp Tyr      50 55 60 Ala Glu Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr  65 70 75 80 Asn Thr Ala Tyr Met Gly Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Val Lys Gly Pro Arg Tyr Tyr Gly Thr Asp Ser Tyr             100 105 110 Tyr Leu Glu Lys Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala         115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser     130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly                 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu             180 185 190 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr         195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg     210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr         275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln             340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu         355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu                 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val             420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln         435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr     450 455 460 Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr 465 470 475 480 Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His                 485 490 495 Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys             500 505 510 Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu         515 520 525 Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys     530 535 540 Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys 545 550 555 560 Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys                 565 570 575 Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp             580 585 590 Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser         595 600 605 Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile     610 615 620 Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 625 630 635 <210> 37 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> D2 heavy chain + TNFR2 <400> 37 Asp Val His Ser Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys   1 5 10 15 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr              20 25 30 Phe Thr Asn Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly          35 40 45 Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Asn Thr Gly Tyr      50 55 60 Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Met Ser Thr  65 70 75 80 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Arg Gly Leu Pro Trp Gly Glu Asn Gly Leu Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys         115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly     130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr                 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val             180 185 190 Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn         195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro     210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp                 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp             260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly         275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn     290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro                 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu             340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn         355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile     370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys                 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys             420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu         435 440 445 Ser Leu Ser Pro Gly Lys Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala     450 455 460 Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr 465 470 475 480 Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val                 485 490 495 Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser             500 505 510 Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly         515 520 525 Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu     530 535 540 Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser 545 550 555 560 Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro                 565 570 575 Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys             580 585 590 Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp         595 600 605 Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn     610 615 620 Ala Ser Met Asp Ala Val Cys Thr 625 630 <210> 38 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 heavy chain variable region <400> 38 Glu Tyr Ala Phe His   1 5 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 heavy chain variable region <400> 39 Asp Phe Thr Pro Ala Phe Gly Glu Gly Asp Thr Ala Gln Lys Phe Gln   1 5 10 15 Gly     <210> 40 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 heavy chain variable region <400> 40 Glu Thr Ala Ser Thr Tyr   1 5 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A3 light chain variable region <400> 41 Thr Gly Pro Thr Ile Gly Ala Gly Tyr Asp Val His   1 5 10 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A3 light chain variable region <400> 42 Gly Asn Leu Asn Arg Pro Ser   1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A3 light chain variable region <400> 43 His Thr Tyr Asp Ser Ser Leu Ser   1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 heavy chain variable region <400> 44 Asp Tyr Ala Met His   1 5 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 heavy chain variable region <400> 45 Val Ile Ser Gly Asp Asp Gly Thr Thr His Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 46 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 heavy chain variable region <400> 46 Asp Met Tyr Pro Tyr Gly Asp Tyr Gly Tyr Leu Gln His   1 5 10 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of A10 light chain variable region <400> 47 Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala   1 5 10 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of A10 light chain variable region <400> 48 Ala Ala Ser Thr Leu Gln Ser   1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of A10 light chain variable region <400> 49 Gln Gln Leu Asn Thr Tyr Pro Leu   1 5

Claims (16)

(a) a promoter;
A monoclonal antibody specific for IL-6R (Interleukin-6 receptor) and a TNFR2 (Tumor necrosis factor receptor type 2) protein linked to the C-terminus of the constant region of the monoclonal antibody or a fragment comprising the extracellular region of the TNFR2 A gene encoding a bispecific protein specific for IL-6R and TNF-a; And
A gene expression cassette comprising a transcription terminator,
(b) a site-specific recombination region located outside the gene expression cassette, and
(c) does not include the cloning start point and selectable marker gene
As a non-viral vector,
The IL-6R-specific monoclonal antibody may be,
A heavy chain variable region comprising the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 and the heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprising the light chain CDR1 A light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6;
A heavy chain variable region comprising the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, the heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and the heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 9 and the light chain variable region comprising the light chain CDR1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 12;
A heavy chain variable region comprising the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, the heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 14 and the heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprising the light chain CDR1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 18; And
A heavy chain variable region comprising the heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 19, the heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 20 and the heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 21, and the light chain variable region comprising the light chain CDR1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 24
, &Lt; / RTI &gt; which is a monoclonal antibody selected from the group consisting of &
The TNFR2 protein or a fragment comprising the extracellular domain of the TNFR2 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 33 or a polypeptide comprising N-terminal 23 to 179 of the amino acid sequence of SEQ ID NO: 33,
Wherein said site-specific recombination region is attB, attP, attR or attL from Escherichia coli or bacteriophage lambda,
A pharmaceutical composition for the prevention or treatment of rheumatoid arthritis comprising a non-viral vector.
delete The composition of claim 1, wherein said IL-6R-specific monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.
delete The composition of claim 1, wherein said IL-6R-specific monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28.
delete The composition of claim 1, wherein said IL-6R-specific monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
delete The composition of claim 1, wherein said IL-6R-specific monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32.
delete delete 2. The composition of claim 1, wherein the promoter is a CMV (cytomegalovirus) promoter.
2. The composition of claim 1, wherein the terminator comprises an SV40 polyadenylation sequence.
delete 2. The composition of claim 1, wherein the non-viral vector is double stranded DNA in the form of a circular supercoil.
delete

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